When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This recombinant lacked 10593 bp of sequence information, located between 13

Direct evidence of in vivo apoptosis of Spodoptera litura larvae was demonstrated by haemocoel inoculation with wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virus (BV). In sharp contrast to natural infection, cadavers did not melt, liquefy and melanize. Typical morphological changes of apoptosis in insect haemocytes post-infection, including blebbing of the cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, were observed by light and electron microscopy. Total DNAs extracted from virus-infected haemocytes showed DNA ladders. Cleavage of chromatin DNA by endogenous endonucleases were detected in the cells of most tissues cells, including epithelial cells and fat body cells, using terminal dUTP nick end labelling assays. Virogenic stroma and viral nucleocapsids could be seen in the nuclei of a few haemocytes. Yields of BV and OV (occluded virus) produced from the infected S. litura larvae were much lower than from the infected S. exigua larvae. These data suggest that host apoptotic responses to virus infection reduce AcMNPV spread at the level of the organism and that apoptosis could be host-range limiting factor for baculovirus infections.

A new species of entomopathogenic nematode, Steinernema guangdongense sp. n. was recovered from a soil sample collected from Jijia town in the western part of Guangdong province, the Peoples Republic of China during a survey for entomopathogenic nematodes in 2001. The nematode can be separated from other described species of Steinernema, by morphological, morphometrical characteristics of different stages of the nematode, by crossbreeding tests and by characterizations and phylogeny of DNA sequences of either a partial 28S or the internal transcribed spacer regions of rDNA. This nematode is closest to S. longicaudum. It can be distinguished from that nematode by characteristics of different stages. For infective juveniles, although the body length is almost similar (1055 μm compared to 1063μm), body diameter of the new species is larger; values of EP (length from anterior end to excretory pore), NR (length from anterior end to nerve ring) and a body length/body width ratio are smaller, and tail with dorsal constriction. For male, the new species has longer spicule, not well curved, spicule head shorter, shaft not prominent or absent and spicule tip not suddenly tapered as shown in S. longicaudum. Also, the ratios SW (spicule length/anal body width) and GS (gubernaculums/spicule) are smaller. For female, the presence of a small double flapped epiptygma, a small projection on dorsal side of the tail tips and prominent post-anal swelling is typical for the new species.

The GP37 amino acid sequence of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) was compared with other baculovirus GP37, entomopoxvirus fusolin, the enhancing factor of Pseudaletia separata entomopoxvirus, and Alteromonas sp. chitin-binding protein 1. In these proteins, five 'conserved regions' previously reported constitute a chitin-binding domain. SpltMNPV GP37 effectively bound to purified crab shell chitin and the dissociation constant (Kd) for binding was 0.28 mM. Immunofluorescence analysis indicated that SpltMNPV GP37 was located in both cytoplasm and nucleus. Immunoblot analysis revealed that this protein was present in the envelopes of both occlusion body-derived virus and budded virus. Further analysis suggested that GP37 may bind to the chitin component of the peritrophic membrane of S. litura larvae.